This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The overall goal of this project is to understand how members of the Pestivirus family assemble, and how they enter the host cell. We focus on a key step in the entry of enveloped viruses into the cell: fusion of the viral and host cell membranes. Membrane fusion is an essential step in entry of enveloped viruses because it delivers the viral genome into the cytoplasm. We have previously determined the pre- and postfusion structures of the postfusion of several envelope proteins from the related flavivirus family. These structures yielded important insights into the membrane fusion mechanism of these viruses. However, flaviviruses and pestiviruses have substantially divergent envelope proteins, and the pestivirus envelope proteins are expected to adopt novel protein folds. The mechanism of membrane fusion may also differ in significant ways. There is currently no structural information of any pestivirus structural protein (only structure of the so-called non-structural proteins are available). In order to gain a better understanding of pestivirus assembly and cell entry, we propose to determine the structure of three different hepacivirus envelope proteins. Our structures of pestivirus envelope proteins will advance our understanding of pestivirus assembly and cell entry. Pestiviruses cause serious damage to livestock and are related to important human pathogens such as Hepatitis C virus. Our work will provide the first structural insight into the proteins that form the outer protein shell of pestiviruses. A direct view of this outer protein shell is essential for understanding how pestiviruses assemble their protein coats, and will reveal the precise nature and extent of the protein epitopes that are exposed on the viral surface. This will provide a framework for the rational design of antiviral compounds that viral membrane fusion, or viral attachment to the cell.